This invention relates to the sialyltransferase gene family, a group of glycosyltransferases responsible for the terminal sialylation of carbohydrate groups of glycoproteins, glycolypids and oligosaccharides which contain a conserved region of homology in the catalytic domain. Members of the sialyltransferase gene family comprise Gal.beta.1,3GalNAc .alpha.2,3 sialyltransferase and Gal1,3(4)GlcNAc .alpha.2,3 sialyltransferase. The invention further relates to novel forms and compositions thereof and particularly to the means and methods for the identification and production of members of the sialyltransferase gene family to homogeneity in significant useful quantities. This invention also relates to preparation of isolated deoxyribonucleic acid (DNA) coding for the production of sialyltransferases; to methods of obtaining DNA molecules which code for sialyltransferases; to the expression of human and mammalian sialyltransferases utilizing such DNA, as well as to novel compounds, including novel nucleic acids encoding sialyltransferases or fragments thereof. This invention is also directed to sialyltransferase derivatives, particularly derivatives lacking cytoplasmic and/or transmembrane portions of the protein, and their production by recombinant DNA techniques.
Sialyltransferases are a family of enzymes that catalyze the transfer of sialic acid (SA) to terminal portions on the carbohydrate groups of glycolipids and oligosaccharides in the general reaction:
Cytididine 5 monophosphate-sialic acid (CMP-SA)+HO-acceptor.fwdarw.CMP+SA-O-Acceptor (Beyer, T. A. et. Adv. Enzynol. 52, 23-175 1981!). Sialyltransferases are found primarily in the Golgi apparatus of cells where they participate in post-translational glycosylation pathways. (Fleischer, B. J. Cell Biol. 89, 246-255 1981!). They are also found in body fluids, such as breast milk, colostrum and blood. At least 10-12 different sialyltransferases are required to synthesize all the sialyloligossacharide sequences known. Four sialyltransferases have been purified. (Weinstein, J. et al., J. Biol. Chem. 257, 13835-13844 1982!; Miagi, T and Tsuiki, S. Eur. J. Biochem. 125, 253-261 1982!; and Joziasse, D. H. et al., J. Biol. Chem. 260, 4941-4951 1985!). More specifically, a Gal.beta.1,4GlcNAc .alpha.2-6 sialyltransferase and a Gal.beta.1,3(4)GlcNAc .alpha.2-3 sialyltransferase have been purified from rat liver membranes. (Weinstein et al. Ibid)
Other glycosyltransferases have been isolated as soluble enzymes in serum, milk or colostrum including sialyl-, fucosyl-, galactosyl-, N-acetylgucosaminyl-, and N-acetylgalactosaminyltransferases. (Beyer et al. Ibid.) Bovine and human .beta.-N-acetylglucosamide .beta.1,4-galactosyltransferase has been isolated (Narimatsu, H. et al. Proc. Nat. Acad. Sci. U.S.A. 83, 4720-4724 1986!; Shaper, N. L. et al., Proc. Nat. Acad. Sci. U.S.A. 83, 1573-1577 1986!; Appert, H. E. et al., Biochem. Biophys. Res. Common 139, 163-168 1986!; and, Humphreys-Beyer, M. G. et al., Proc. Nat. Acad. Sci. U.S.A. 83, 8918-8922 1986!. These purified glycosyltransferases differ in size which may be due to the removal of portions of the protein not essential for activity, such as the membrane spanning domains.
Comparison of the deduced amino acid sequences of the cDNA clones encoding the glycosyltransferases including galactosyltransferases, sialyltransferase, fucosyltransferase and N-acetylgalactosaminyltransferase, reveals that these enzymes have virtually no sequence homology. Some insight into how this family of glycosyltransferases might be structurally related has come from recent analysis of the primary structures of cloned sialyltransferases (Weinstein, J. et al., ibid.). However, they all have a short NH.sub.2 -terminal cytoplasmic tail, a 16-20 amino acid signal-anchor domain, and an extended stem region which is followed by the large COOH-terminal catalytic domain Weinstein, J. et al., J. Biol. Chem. 262, 17735-17743 1987! Paulson, J. C. et al., J. Biol. Chem. 264, 17615-17618 1989!. Signal-anchor domains act as both uncleavable signal peptides and as membrane-spanning regions and orient the catalytic domains of these glycosyltransferases within the lumen of the Golgi apparatus. Common amino acid sequences would be expected within families of glycosyltransferases which share similar acceptor or donor substrates; however, surprisingly few regions of homology have been found within the catalytic domains of glycosyltransferases, and no significant sequence homology is found with any other protein in GenBank (Shaper, N. L. et al., J. Biol Chem 216, 10420-10428 1988!, D'Agostaso, G. et al., Eur J. Biochem 183, 211-217 1989! and Weinstein, J. et al., J. Biol. Chem 263, 17735-17743 1987!). This is especially surprising for the Gal .alpha.1,3-GT and GlcNAc .beta.1,4-GT, two galactosyltransferases. However, while these galactosyltransferases exhibit no overall homology, there is a common hexapeptide KDKKND for the Gal .alpha.1,3-GT (bovine, 304-309) and RDKKNE for the GlcNAc .beta.1,4-GT (bovine, human, murine amino acids 346-351). (Joziasse et al., J. Biol Chem 264, 14290-14297 1989!.)
Sialic acids are terminal sugars on carbohydrate groups present on glycoproteins and glycolipids and are widely distributed in animal tissues (Momol, T. et al., J. Biol. Chem. 261, 16270-16273 1986!). Sialic acids play important roles in the biological functions of carbohydrate structures because of their terminal position. For instance, sialic acid functions as the ligand for the binding of influenza virus to a host cell (Paulson, J. C., The Receptors, Vol. 2, Conn, P. M., ed., pp. 131-219, Academic Press 1985!). Even a change in the sialic acid linkage is sufficient to alter host specificity (Roger, G. N. et al., Nature 304, 76-78 1983!). The neural cell adhesion molecule (NCAM) is subject to developmentally regulated polysialylation which is believed to modulate NCAm mediated cell adhesion during the development of the nervous system (Rutishauser, U. et al., Science 240, 53-37 1988! and Rutishauser, U., Adv. Exp. Med. Biol. 265, 179-18 1990!). Recently, a carbohydrate structure, sialyl lewis X (SLe.sup.X) has been shown to function as a ligand for the endothelial leucocyte adhesion molecule ("E-Selectin") which mediates the binding of neutrophils to activated endothelial cells (Lowe et al., 1990; Phillips et al., 1990; Goelz et al., 1990; Walz et al., 1990; Brandley et al., 1990). P-selectin (platelet activation dependent granule to external membrane protein; CD62), another member of the selectin family (Stoolman, L. M., Cell 56, 907-910 1989!), has also been demonstrated to recognize SLe.sup.X present on monocytes and PMNs (Larsen et al., Proc. Natl. Acad. Sci. USA 87, 6674-6678 1990!; Momol et al., J. Biol. Chem. 261, 16270-16273 1986!; Polley et al., Proc. Natl. Acad. Sci. USA 88, 6224-6228 1991!; Chan, K. F. J., J. Biol. Chem. 263, 568-574 1988!; Beyer, T. A. et al., Adv. Enzymol., 52, 23-175 1981!). In both instances, sialic acid is a key component for the carbohydrate structure to function as a ligand. In addition to playing a role in cell adhesion, sialic acid containing carbohydrate structures have been implicated as playing a direct role in differentiation. The hematopoietic cell line HL-60 can be induced to differentiate by treatment with the glycolipid G.sub.M3. Gangliosides are also thought to play a role in modulation of growth factor-protein kinase activities and in the control of the cell cycle.
One such sialyltransferase has been purified, as described in U.S. Pat. No. 5,047,335. While some quantities of purified sialyltransferase have been available, they are available in very low amounts in part because they are membrane bound proteins of the endoplasmic reticulum and the golgi apparatus. Significant cost, both economic and of effort, of purifying these sialyltransferases makes it a scarce material. It is an object of the present invention to isolate DNA encoding sialyltransferase and to produce useful quantities of mammalian, particularly human, sialyltransferase using recombinant DNA techniques. It is a further object to provide a means for obtaining the DNA encoding other members of the sialyltransferase gene family from various tissues as well as from other species. It is a further object of the present invention to prepare novel forms of sialyltransferases. It is still another object herein to provide an improved means for catalyzing the transfer of sialic acid to terminal positions on certain carbohydrate groups. These and other objects of this invention will be apparent from the specification as a whole.